| 摘要: |
| 【目的】探索重叠延伸PCR法在昆虫化学感受蛋白基因点突变上的应用，研究半胱氨酸（Cys32）对小菜蛾（Plutella xylostella）化学感受蛋白1（PlxyCSP1）结合特性的影响。【方法】 用计算机软件模拟PlxyCSP1上Cys32突变前后的蛋白三级结构以及与配体化合物的结合情况，确定点突变位点。根据扩增得到的PlxyCSP1序列设计并合成重叠引物，利用重叠延伸PCR法，将PlxyCSP1上的Cys32突变成色氨酸（Trp32），获得突变体PlxyCSP1-M。将突变后的基因与载体pET-32a(+)连接，转化大肠杆菌BL21(DE3)。【结果】 分子对接图显示，配体可以顺利进入PlxyCSP1的结合口袋，而无法进入PlxyCSP1-M的结合口袋。结合能预测显示，配体化合物与PlxyCSP1的结合能均为正值，与PlxyCSP1-M的结合能均为0。突变后的重组质粒测序结果表明，Cys32（TGC）已突变为Trp32（TGG），经IPTG诱导，该菌表达了分子质量约为36 ku的融合蛋白。【结论】 Cys32是影响PlxyCSP1结构和功能的重要氨基酸残基。Cys32突变为Trp32后，PlxyCSP1-M结合特性发生了变化，不能与配体化合物正常结合。 |
| 关键词: 小菜蛾 化学感受蛋白1 重叠延伸PCR 原核表达 同源建模 分子对接 |
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| 基金项目:国家自然科学基金项目(31071713)；教育部高等学校博士学科点专项科研基金项目(20094404110019) |
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| Overlap extension PCR based site directed mutant of PlxyCSP1 gene in Plutella xylostella |
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| Abstract: |
| 【Objective】This study explored overlap extension PCR based site-directed mutant of insects chemosensory and studied the effects of the cysteine(Cys32) in binding characteristics of PlxyCSP1.【Method】The tertiary structure and binding characteristics of PlxyCSP1 and PlxyCSP1-M were simulated by computer software in order to confirm the mutation site.Overlapping primes were designed according to the sequence of PlxyCSP1 to obtain PlxyCSP1-M which the Cys32 was mutated to the Trp32.PlxyCSP1-M was linked with pET-32a(+) and transformed into E.coli BL21(DE3).【Result】The result of molecular docking displayed that ligand compounds could easily enter binding pocket of PlxyCSP1 but could not enter the binding pocket of PlxyCSP1-M.The binding energies of ligand compounds and PlxyCSP1 were all positive while the binding energies of ligand compounds and PlxyCSP1-M were zero. Recombinant plasmid sequencing showed that the Cys32 was mutated to Trp32 in PlxyCSP1-M and the recombinant strain could express fusion protein which was 36 ku by IPTG induction.【Conclusion】The Cys32 is important to the structure and function of PlxyCSP1.Binding characteristics of PlxyCSP1-M were changed and could not bind with ligand compounds. |
| Key words: Plutella xylostella PlxyCSP1 overlap extension PCR prokaryotic expression homology modeling molecular docking |
